Methods for production of proteins

ABSTRACT

The current invention provides methods for producing a polypeptide as inclusion bodies in bacterial host cells. The present methods are carried out by forming a gene construct comprising the genetic sequence encoding a polypeptide operatively linked to that of an inclusion partner protein, such that host cells comprising the gene construct produce the polypeptide as intracellular inclusion bodies thereby facilitating rapid isolation and purification of recombinant proteins. The invention further provides methods for producing protein molecular weight ladders for us in protein gel electrophoresis, as well as proteins and protein molecular weight ladders produced by these methods.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/034,658, filed Jan. 8, 1997, the contents of which are entirelyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention is in the fields of molecular biology and proteinengineering. The invention is directed to methods for the production ofrecombinant proteins. More specifically, the invention is directed tomethods for producing recombinant proteins as inclusion bodies inbacteria, particularly Escherichia coli. The present invention alsoprovides plasmids, vectors and host cells to be used in the presentinvention for production of recombinant proteins, and methods ofpurification of the proteins produced by these methods. The invention isalso directed to proteins produced by these methods. The invention isalso directed to methods for production of protein molecular weightmarker ladders, and to ladders produced by these methods.

2. Related Art

With the advent of recombinant DNA technology, it has become almostroutine to produce large amounts of proteins in heterologous expressionsystems, such as transformed host cells, for commercial and basicresearch purposes. Among the expression host systems, E. coli is themost popular system because of ease with which E. coli can bemanipulated. However, expression of heterologous proteins in host cellshas some limitations. These include: inefficient translation of mRNA dueto the presence of infrequently used codons (Kane, J., Current Opin.Biotech 6:494-500 (1995)), instability of mRNA in E. coli (Bachmair, A.et al., Science 234:179-186 (1986); Olins, P. & Lee, S., Current Opin.Biotech 6:501-506 (1993)), toxic effect of the protein being expressed(Brosius, J., Gene 27:161-172 (1984); Studier, W. & Mofatt, B., J. MolBiol. 189:113-130 (1986)), and formation of inclusion bodies because ofinappropriate folding of the protein (Schein, C., Bio/Technology7:1141-1149 (1989); Mitraki, A. & King, J., Bio/Technology 7:690-697(1989)). To solve these problems, a variety of techniques have beendeveloped.

Gene fusion is one of the most popular strategies to express proteins ofinterest. This particular technique is used to produce large amounts ofheterologous protein by fusing the protein of interest to the carboxyterminal end of a fusion partner (LaVallie, E., and McCoy, J., Curr.Opin. Biotech 6:501-506). As an example of this approach, methods havebeen developed for selective isolation of a desired protein orpolypeptide by constructing a recombinant vector containing a DNAsequence coding for the desired protein or polypeptide which isoperatively linked to a DNA sequence coding for protein A (WO 84/03103).The expressed fusion protein is then selectively isolated by adsorptiononto an IgG-supporting carrier, which binds protein A, followed bydesorption of the fusion protein. The fusion protein is then cleaved ata unique cleavage site with a cleavage agent, which may includeproteases, hydroxylamine, cyanogen bromide or formic acid, to give thepurified protein.

Most systems used for the manufacture of recombinant polypeptidesattempt to minimize the production of the polypeptide in inclusionbodies in the expressing host cells. One important reason for theseattempts is that the production of polypeptides in inclusion bodiesoften yields a biochemically inactive, denatured, or otherwisefunctionally or structurally compromised polypeptide upon its releasefrom the inclusion bodies via standard solubilization techniques. Whilea variety of methods have shown some promise in minimizing inclusionbody formation, gene fusion techniques in particular have been utilizedto produce soluble proteins which otherwise would have been produced asinclusion bodies.

However, the formation of inclusion bodies within host cells can also beadvantageous. For example, inclusion bodies constitute highly dense andconcentrated “packets” of the desired polypeptide, from whichcontaminating host cell proteins can be removed by methods as simple ascentrifugation. After their isolation, controlled conversion of theinclusion bodies to a soluble form could provide a rich source of thedesired polypeptide in its pure, biologically active or structurallyintact form. The difficulty with such an approach, however, has beenthat it is usually nearly impossible to predict whether or not arecombinant polypeptide will form inclusion bodies when it is expressedin a host cell.

Thus, the current invention provides a system in which controlledformation of inclusion bodies is used to produce a desired polypeptide.By this controlled formation of inclusion bodies, purification of thedesired polypeptide is rendered faster and more complete, and subsequentcontrolled solubilization of the inclusion bodies provides a high yieldof pure polypeptide in its active form.

BRIEF SUMMARY OF THE INVENTION

The current invention provides a system wherein the genetic sequenceencoding a first polypeptide is operatively linked or fused to thatencoding an inclusion partner protein, such as thioredoxin or a modifiedthioredoxin, which is capable of forming inclusion bodies in a host cellupon expression. Specifically, the invention provides a method forproducing a polypeptide in the form of inclusion bodies comprising (a)obtaining a host cell comprising a first nucleic acid molecule encodinga recombinant polypeptide operatively linked to a second nucleic acidmolecule encoding an inclusion partner protein, thereby forming a genefusion construct; and (b) cultivating the above host cell underconditions favoring production of the polypeptide as inclusion bodies inthe host cell. The invention also provides the above method furthercomprising (c) isolating the inclusion bodies from the host cell; and(d) releasing the polypeptide from the inclusion bodies. According tothe present invention, the first nucleic acid molecule encoding thepolypeptide may be obtained from a prokaryotic cell, particularly abacterial cell and most particularly an Escherichia coli cell, or from aeukaryotic cell, particularly an animal cell, a plant cell or a yeastcell, more particularly a mammalian animal cell, and most particularly ahuman cell, and the second nucleic acid molecule encoding the inclusionpartner protein may be obtained from a bacterial cell, most preferablyan Escherichia coli cell. The inclusion partner protein may be anyprotein that forms an inclusion body upon expression in a host cell, andis preferably a bacterial protein, more preferably a bacterialthioredoxin or modified bacterial thioredoxin, and most preferably acarboxy terminal-truncated form of E. coli thioredoxin. Preferably, thegene fusion construct is inserted into a vector prior to beingintroduced into the host cell. According to one aspect of the invention,the polypeptide of interest may be released from inclusion bodies,formed by the gene fusion construct, by cleavage with a chemical such ascyanogen bromide, or more preferably with an enzyme such as thrombin orenterokinase. According to another aspect, a nucleic acid sequenceencoding a protein-specific cleavage site may be placed between thenucleic acid sequence encoding the inclusion partner protein and therecombinant polypeptide in the gene fusion construct; upon expression ofthe fusion protein as inclusion bodies in the host cells, therecombinant polypeptide may then be released therefrom by treating theinclusion bodies with an enzyme or other chemical that specificallyrecognizes and cleaves at the protein-specific cleavage site. Theinvention also provides the above-described methods wherein the genefusion construct comprises plasmid pTrcprl-monomer, and provides plasmidpTrcprl-monomer. The invention is also directed to the above-describedmethods wherein the host cell is a bacterial cell, most preferably anEscherichia coli cell, and wherein the vector used is an expressionvector, most preferably plasmids pTrc99A or pTrxfus. The invention alsoprovides these vectors, and host cells, particularly bacterial cells andmost particularly Escherichia coli cells, comprising these vectors.Although the present invention is most particularly directed to methodsfor the production of fragments of the gene 32 protein of bacteriophageT4, of KpnI methylase and Dead-Box protein, any recombinant polypeptidemay be produced by the present methods. The invention also providesrecombinant polypeptides produced by the above-described methods. Thus,the present system provides reliable methods for producing anyheterologous protein as inclusion bodies in a host cell, therebyfacilitating the rapid isolation and purification of recombinantproteins produced in bacterial host cells. In addition, the methodsprovided by the present invention may be used to produce polypeptidesthat are small or difficult to express, as well as those that are toxicto host cells such as E. coli.

The invention also provides methods for producing a protein molecularweight marker ladder, comprising (a) obtaining one or more nucleic acidmolecules wherein each of the nucleic acid molecules encodes one or morepolypeptides of different molecular weights of the molecular weightladder; (b) transforming one or more host cells with one or more of thenucleic acid molecules; (c) culturing the host cells under conditionsfavoring the production of each of the polypeptides of the molecularweight ladder; and (d) isolating each of the polypeptides. The inventionis particularly directed to such methods wherein at least one of thenucleic acid molecules encodes a plurality of the polypeptides ofdifferent molecular weights of the molecular weight ladder, and whereinthe nucleic acid molecules each encode a different polypeptide of themolecular weight ladder. The invention is also directed to such methodswherein the host cell comprises a nucleic acid molecule encoding aplurality of polypeptides of the molecular weight ladder, and whereineach of the host cells comprises a different nucleic acid molecule eachencoding a different polypeptide of the molecular weight ladder. Theinvention also provides such methods wherein a host cell comprises twoor more of the nucleic acid molecules each encoding a differentpolypeptide of the molecular weight ladder, and wherein such methodfurther comprises admixing each of the different polypeptides to form amolecular weight ladder. The present invention is particularly directedto such methods wherein the polypeptides of the molecular weight ladderare produced as inclusion bodies, and wherein the nucleic acid moleculeencoding the polypeptide(s) is inserted into a vector, most preferablyan expression vector, prior to transforming the host cells. Proteinmolecular weight ladders produced by the methods of the presentinvention are preferably prestained, and the invention provides optimalconditions for prestaining of the proteins to produce these molecularweight ladders. The present invention also provides protein molecularweight marker ladders, which are preferably prestained, produced bythese methods.

The invention also generally relates to methods for producing a stainedprotein and more particularly prestained protein ladders. Such methodsof the invention comprise contacting the one or more proteins orpolypeptides of interest with one or more dyes under conditionssufficient to completely or substantially completely label or complexthe dye(s) to the protein molecule(s). Preferably, the staining methodof the invention is performed on the proteins or protein sample prior tosize separation by, for example, gel electrophoresis. Thus, use of theprotein or polypeptide staining method of the invention provides ahomogeneous or near homogeneous sample in which all or substantially allof the proteins or polypeptides in the sample have been stained orcomplexed with the dye of interest. Such uniform staining providesincreased color intensity upon examination of stained proteins due atleast in part to more dye being complexed with the proteins orpolypeptides (e.g., increased staining of the proteins of interest).Additionally, because of the uniformity and/or completeness of staining,the character of the stained protein will appear more consistent insubsequent analysis. Thus, when performing size analysis on the stainedproteins or polypeptides of the invention, the proteins or polypeptideswill be the same or substantially the same size. Such a feature of thestained proteins or polypeptides of the invention provides for superiorprotein molecular weight markers which allow more accurate sizedetermination of an unknown protein or polypeptide.

The invention thus relates to a method of staining one or morepolypeptides or proteins comprising:

(a) mixing or contacting a sample comprising the one or morepolypeptides or proteins with one or more dyes; and

(b) incubating the mixture under conditions sufficient to producestained proteins or polypeptides having the same or substantially thesame size. Such method may further comprise separating the stainedproteins by size. Size separation may be accomplished by any knowntechnique, including gel electrophoresis, capillary electrophoresis, gelfiltration chromatography and the like.

The invention also relates to a method for staining one or morepolypeptides or proteins comprising:

(a) mixing or contacting a sample comprising the one or morepolypeptides or proteins with one or more dyes; and

(b) incubating the mixture under conditions sufficient to producestained proteins or polypeptides wherein substantially all of theproteins or polypeptides are complexed with the dye. Such methods mayfurther comprise separating the stained proteins by size using standardtechniques such as those described above.

Any conditions may be utilized to produce the desired result inaccordance with the invention. In particular, protein concentrations,dye concentrations, pH, ionic conditions, temperature, and duration ofexposure, or combinations of these parameters, may be varied to producestained proteins or prestained molecular weight markers of theinvention. In accordance with the invention, pH of the solution to whichthe protein(s) and dye(s) are added may be varied from about 7-12,incubation temperature may be varied between about 20° C.-80° C. (morepreferably about 37° C.-70° C., and still more preferably about 50°C.-70° C.), and the duration of incubation may vary from about 1-200hours (preferably about 2-200 hours, about 2-100 hours, about 6-100hours, about 6-72 hours, about 6-48 hours, more preferably about 12-48hours, and still more preferably about 12-24 hours).

Any one or a number of proteins or peptides may be stained in accordancewith the invention. Such staining methods may be accomplished ondifferent proteins (different size and/or type) at the same time orseparately. If desired, separately stained proteins may be mixed afterstaining to provide a mixture of stained proteins having different sizesto produce, for example, a protein molecular weight ladder of theinvention. Preferably, the molecular weight ladder of the inventioncomprises at least two and preferably at least three proteins ofdifferent sizes. More preferably, the ladders of the invention comprise3-20, still more preferably 3-15, and still more preferably 3-10,proteins of different sizes.

The invention also relates to a method for sizing one or more proteinsor polypeptides of unknown size or molecular weight, comprising:

(a) separating, according to size, the protein molecular weight ladderof the invention, and the one or more proteins or polypeptides ofunknown size; and

(b) determining the size and/or molecular weight of the protein(s) orpolypeptide(s). Such determination may be made by comparison of themobility of the unknown protein(s) or polypeptide(s) to that of themolecular weight ladder of the invention by standard techniques such asgel electrophoresis, capillary electrophoresis, etc.

The invention also provides for stained polypeptides and stainedmolecular weight markers produced in accordance with the methods of theinvention and to kits containing them. Such kits comprise a carriermeans, such as a box, carton, or the like, being compartmentalized toreceive in close confinement therein one or more container means such astubes, vials, ampules, bottles or the like, wherein a first containermeans comprises one or more stained polypeptides of the invention or oneor more of the stained molecular weight marker ladders of the invention.In one such aspect of the invention, a number of individual containersmay be provided in a kit, each container containing a different sized(and/or type) stained polypeptide, such that the end user mayselectively prepare different molecular weight markers having adifferent combination of differently sized proteins. Thus, the inventionprovides the end user with flexibility in making an appropriate markerladder depending on the need. Moreover, kits of the invention may alsoprovide separate containers containing differently stained polypeptides(e.g., stained with different dyes), thus providing the end user withflexibility not only in varying the size or pattern of the molecularweight ladder but also the color or colors attributed to the individualpeptides or bands in the ladder. The kits of the invention may furthercomprise one or more additional container means containing componentswhich facilitate size analysis of proteins, such as acrylamide, SDS, gelor capillary electrophoresis reagents and/or equipment, and the like.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 (SEQ ID NO:1) is a depiction of the 264-bp (gene32) AvaI fragmentderived from pPrL2107 used to make multimers in pPrL2001.

FIG. 2 (SEQ ID NO:2) is a depiction of the 261-bp fragment with a singleAvaI site used to prepare plasmid ptcprl-monomer.

FIG. 3 (SEQ ID NO:5) is a depiction of the delta thioredoxin sequence,plasmid pTrxfusprl10A, used to make the 10 kD protein.

FIG. 4 (SEQ ID NO:8) is a depiction of the trxA-concat sequence havingNcoI and NdeI sites used to make concatamers. This plasmid, designatedpTrxA-concat, served as the inclusion partner.

FIG. 5 (SEQ ID NO:11) is a depiction of the delta thioredoxin sequenceused to make trxAtrxA concatamers.

FIG. 6 (SEQ ID NO:14) is a depiction of the 138-bp Dead-box fusionpartner fragment used to make the molecular weight ladder by fusion withpTrxA-concat.

FIG. 7 (SEQ ID NO:17) is a depiction of the 15 kD KpnI methylase fusionpartner fragment used to make the molecular weight ladder by fusion withpTrxA-concat.

FIG. 8 is a color photograph of a 4-20% SDS-PAGE gradient gel of fourdifferent load volumes of the prestained molecular weight markers,demonstrating the 50 kD reference band stained with eosin isothiocyanate(pink band) and the remaining bands in the ladder stained with RBBR(blue bands).

FIG. 9 is a photograph of a 4-20% SDS-PAGE gradient gel of 50 kD (lanes1-8) and 60 kD (lanes 9-13) reference proteins prestained overnight witheosin isothiocyanate at room temperature (lanes 1-4 and 9-12) or at 50°C. (lanes 5-8 and 13) at the indicated pHs. M: molecular weight standardladders (two different preparations).

FIG. 10 is a photograph of a 4-20% SDS-PAGE gradient gel of 40 kD (lanes1-12) and 50 kD (lanes 13, 14) molecular weight markers prestainedovernight with eosin isothiocyanate at room temperature (lanes 1-3, 7-9)or at 50° C. (lanes 4-6, 10-14) at the indicated pHs. M: molecularweight standard ladder.

FIG. 11 is a photograph of a 4-20% SDS-PAGE gradient gel of 50 kDmolecular weight markers prestained overnight with eosin isothiocyanateat room temperature (lanes 1-3, 7-9) or at 50° C. (lanes 4-6, 10-14) atthe indicated pHs. M: molecular weight standard ladder.

FIG. 12 is a photograph of a 4-20% SDS-PAGE gradient gel of 30 kD (lanes1, 5, 12), 40 kD (lanes 2, 7, 10), 50 kD (lanes 3, 8, 11), and 60 kD(lanes 4, 6, 9) molecular weight markers prestained with Procion Red(lanes 1-4) or with eosin isothiocyanate (lanes 5-12) at the indicatedpHs. M: molecular weight standard ladders (two different preparations).

FIG. 13 is a photograph of a 4-20% SDS-PAGE gradient gel of 30 kD (lanes1-2), 40 kD (lanes 3-4), 5010 (lanes 5-6) and 60 kD (lanes 7-8)molecular weight markers prestained with malachite green isothiocyanateat the indicated pHs. M: molecular weight standard ladders (twodifferent preparations).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms conventionally usedin the fields of molecular biology and protein engineering are utilizedextensively. In order to provide a clear and consistent understanding ofthe specification and claims, and the scope to be given such terms, thefollowing definitions are provided.

The term “polypeptide” is used herein to mean a sequence of contiguousamino acids, of any length. As used herein, the terms “peptide” or“protein” may be used interchangeably with the term “polypeptide.”

The term “nucleic acid molecule” as used herein refers to a sequence ofcontiguous nucleotides which may encode a full-length polypeptide or afragment of any length thereof, or may be non-coding.

The term “inclusion partner protein” is used herein to mean any proteinor fragment, portion, derivative or variant thereof, which formsinclusion bodies upon expression in a host cell; nucleic acid moleculesencoding inclusion partner proteins may be fused to those encodingpolypeptides of interest in order to cause the polypeptide of interestto be co-expressed in the form of inclusion bodies in a host cell.

The term “gene fusion construct” as used herein means a nucleic acidmolecule which is the product of the operative linkage or fusion of anucleic acid molecule encoding a polypeptide of interest to a nucleicacid molecule encoding an inclusion partner protein. A gene fusionconstruct as defined herein may include additional nucleic acidsequences comprising expression signals (such as promoters or enhancers)which are recognized by a host cell and which direct the expression ofthe gene fusion construct to produce the polypeptide of interest.

The phrases “substantially all of the polypeptides are complexed with adye” or “substantially all of the polypeptides are stained with a dye”or “substantially all of the polypeptides are labeled with a dye” may beused interchangeably and as used herein mean that substantially all ofthe polypeptides or proteins in a sample have been completely orsubstantially completely complexed, stained, or labeled with one or moredyes. Such completion of staining can be determined by any number ofanalytical techniques, although analysis of mobility and stainingintensity by gel electrophoresis is preferred (see Examples below). Forexample, incomplete or partial staining of a protein sample results in aheterogeneous population of proteins, each of which may have a differentmobility during gel electrophoresis. Upon complete or substantiallycomplete staining, however, mobility will remain substantially unchangedeven upon further staining (i.e., further incubation with dye). Thus,complete or substantially complete staining may be measured by suchmobility changes, or the lack or substantial lack thereof. Alternativelyor in addition to such mobility changes, completion of staining may bedetermined by changes in intensity of staining. Thus, upon complete orsubstantially complete staining in accordance with the invention, thestain intensity of a protein sample of interest, determined, forexample, by gel electrophoresis, will not substantially change uponfurther staining (i.e., further incubation with dye).

Overview

The present invention provides a method for producing and isolatingrecombinant polypeptides from host cells, wherein the recombinantpolypeptides are produced as inclusion bodies in the host cells.Specifically, the method involves (a) obtaining a host cell comprising afirst nucleic acid molecule encoding a recombinant polypeptideoperatively linked to a second nucleic acid molecule encoding aninclusion partner protein, thereby forming a gene fusion construct; and(b) cultivating the above host cell under conditions favoring productionof the polypeptide as inclusion bodies in the host cell. The inventionalso provides the above method further comprising (c) isolating theinclusion bodies from the host cell, most preferably by centrifugation;and (d) releasing the polypeptide from the inclusion bodies. Accordingto the present invention, the first nucleic acid molecule encoding thepolypeptide may be obtained from a prokaryotic cell, particularly abacterial cell and most particularly an Escherichia coli cell, or from aeukaryotic cell, particularly an animal cell, a plant cell or a yeastcell, more particularly a mammalian animal cell, and most particularly ahuman cell, and the second nucleic acid molecule encoding the inclusionpartner protein may be obtained from any cell, preferably a bacterialcell, and most preferably an Escherichia coli cell. The inclusionpartner protein used in the present invention may be any protein thatforms an inclusion body upon expression in a host cell, and ispreferably a bacterial protein, more preferably a bacterial thioredoxinor modified bacterial thioredoxin, and most preferably a carboxyterminal-truncated form of E. coli thioredoxin as described in moredetail below. Preferably, the gene fusion construct is inserted into avector prior to being introduced into the host cell. According to oneaspect of the invention, the polypeptide of interest may be releasedfrom inclusion bodies, formed by the gene fusion construct, by cleavagewith a chemical such as cyanogen bromide, or more preferably with anenzyme such as thrombin or enterokinase. According to another aspect, anucleic acid sequence encoding a protein-specific cleavage site may beplaced between the nucleic acid sequence encoding the inclusion partnerprotein and the recombinant polypeptide in the gene fusion construct;upon expression of the fusion protein as inclusion bodies in the hostcells, the recombinant polypeptide may then be released therefrom bytreating the inclusion bodies with an enzyme or other chemical thatspecifically recognizes and cleaves at the protein-specific cleavagesite. The invention also provides the above-described methods whereinthe gene fusion construct comprises plasmid pTrcprl-monomer, andprovides plasmid pTrcprl-monomer. The invention is also directed to theabove-described methods wherein the host cell is a bacterial cell, mostpreferably an Escherichia coli cell, and wherein the vector used is anexpression vector, most preferably plasmids pTrc99A or pTrxfus. Theinvention also provides these vectors, and host cells, particularlybacterial cells and most particularly Escherichia coli cells, comprisingthese vectors. The invention further provides recombinant polypeptidesmade by the above methods, plasmid pTrcprl-monomer, and plasmidpTrxA-concat.

In another preferred embodiment, the invention provides methods formaking a protein molecular weight ladder, which is preferablyprestained, and a protein molecular weight ladder produced by thesemethods.

Although the present invention is most particularly directed to methodsfor the production of a fragment of the gene 32 protein of bacteriophageT4, KpnI methylase, or Dead-Box protein, it will be readily appreciatedby one of ordinary skill in the art that using the methods of thepresent invention, any polypeptide comprising a sequence of contiguousamino acids of any length may be produced as inclusion bodies in a hostcell and isolated therefrom.

Gene Fusion

The methods of the present invention utilize the technique of genefusion to produce a gene fusion construct comprising the nucleic acidmolecule encoding a first polypeptide operatively linked to a secondnucleic acid molecule encoding an inclusion partner protein. The nucleicacid molecule encoding the first polypeptide may be obtained from abacterial cell, particularly an E. coli cells; from an animal cell,preferably a mammalian cell and most preferably a human cell; a plantcell; or a yeast cell. As described in more detail below, the nucleicacid molecule encoding the inclusion partner protein may be obtainedfrom any cell, preferably from a bacterial cell, and most preferablyfrom an Escherichia coli cell.

Methods for construction of gene fusion constructs comprising a DNAsequence encoding a desired polypeptide, fused to a prokaryotic DNAsequence, are well-known in the art (see, e.g., Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press, pp. 17.2-17.9 (1989);Ausubel, F. M., et al., eds., Current Protocols in Molecular Biology,New York: John Wiley & Sons, Inc., pp. 16.4.1-16.8.14 (1994)). Othersuitable methods that are routine to one of ordinary skill in the artmay also be used equivalently in the methods of the present invention.

Vectors and Host Cells

The present invention also relates to vectors which comprise theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and methods for theproduction of a recombinant polypeptide using these vectors and hostcells.

The vector used in the present invention may be, for example, a phage ora plasmid, and is preferably a plasmid. Preferred are vectors comprisingcis-acting control regions to the nucleic acid encoding the polypeptideof interest. Appropriate trans-acting factors may be supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters. Other suitable promoters will be known to the skilledartisan. The gene fusion constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiation codon at the beginning, and a termination codon(UAA, UGA or UAG) appropriately positioned at the end, of thepolynucleotide to be translated.

The expression vectors will preferably include at least one selectablemarker. Such markers include tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria.

Among vectors preferred for use in the present invention include pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; pcDNA3 available from Invitrogen; and pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available fromPharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Representative examples of appropriate host cells include, but are notlimited to, bacterial cells such as E. coli, Streptomyces spp., Erwiniaspp., Klebsiella spp. and Salmonella typhimurium. Preferred as a hostcell is E. coli, and particularly preferred are E. coli strains DH10Band Stb12, which are available commercially (Life Technologies, Inc;Rockville, Md.).

Inclusion Partners

It has been unexpectedly discovered in the present invention that use ofa modified version of the gene encoding the inclusion partner proteinwill induce the host cell to produce the fusion protein, comprising thepolypeptide of interest, as intracellular inclusion bodies. As usedherein, the term “modified version of a gene” means a version of a genecomprising an alteration of the normal or most commonly encounteredsequence of the gene, which results in the expression of the encodedprotein, in a fused or unfused state, in inclusion bodies in a hostcell. Such alterations may include, but are not limited to, deletions,substitutions, insertions, point mutations, and the like.

Preferred inclusion partner proteins for use in the present inventioninclude, but are not limited to, modified versions of E. colimaltose-binding protein (Betton and Hofnug, J. Biol. Chem. 271:8046-8052(1996)), E. coli RNAse II (Coburn and Mackie, J. Biol. Chem.271:1048-1053 (1996)), E. coli alkaline phosphatase (Derman andBeckwith, J Bacteriol. 177:3764-3770 (1995); Georgiou et al., Appl. Env.Microbial. 52:1157-1161 (1986)), E. coli phospholipase A (Dekker et al.,Eur. J. Biochem. 232:214-219 (1995)), E. coli β-lactamase (Rinas andBailey, Appl. Env. Microbiol. 59:561-566 (1993); Georgiou et al., Appl.Env. Microbiol. 52:1157-1161 (1986)), Salmonella typhimurium MalKprotein (Schneider et al., Prot. Exp. Purif. 6:10-14 (1995)),Clostridium thermocellum endoglucanase D (Tokatlidis et al., FEBS Lett.282:205-208 (1991)), Bacillus thuringiensis subsp. aizawai IPL7insecticidal proteins (Oeda et al., J. Bacteriol. 171:3568-3571 (1989),human procathepsin B (Kuhelj et al., Eur. J. Biochem. 229:533-539(1995)), porcine interferon-γ (Vandenbroeck et al., Eur. J. Biochem.215:481-486 (1993)), T5 DNA polymerase (Chatterjee et al., Gene 97:13-19(1991)), and E. coli thioredoxin (Hoog et al., BioSci. Rep. 4:917-923(1984)). More preferable for use in the present invention is a modifiedE. coli thioredoxin, which as used herein is a thioredoxin proteinhaving the ability to form inclusion bodies (in fused or unfusedconstructs) upon expression in a host cell. Particularly preferred is adeletion mutant of the E. coli thioredoxin encoded by the trxA gene, andmost particularly the carboxy terminal-truncated form of E. coli trxAhaving a nucleotide sequence as set forth in SEQ ID NO:8, hereinafterdesignated pTrxA-concat. The recombinant host cell comprisingpTrxA-concat, E. coli DH10B(pTrxA-concat), was deposited on Jan. 6,1997, with the Collection, Agricultural Research Culture Collection(NRRL), 1815 North University Street, Peoria, Ill. 61604 USA, as DepositNo. NRRL B-21653.

Truncated versions of trxA that may be used in the present inventioninclude those wherein 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50amino acids are deleted from the carboxy terminus of thioredoxin,preferably those wherein 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33amino acids are deleted, and most preferably those wherein 23 aminoacids are deleted (pTrxfusprl10A).

Methods for making and expressing modified gene fusion constructs, suchas a construct encoding a modified or truncated thioredoxin,are-well-known to one of ordinary skill in the art and are amplydescribed in the literature (see, e.g., Winnacker, From Genes to Clones,New York: VCH Publishers, pp. 451-481 (1987)), and in detail in theExamples below. To determine if a particular modified gene fusionconstruct induces the production of inclusion bodies in a host cell, theconstruct may be transferred into a host cell and expressed as describedbelow. A suspension of host cells may then be examined for the presenceof inclusion bodies by any means, such as microscopy (e.g., phasecontrast, Nomarski interference, electron or fluorescence microscopy),suitable for the detection of the presence of inclusion bodies withinindividual host cells.

For use in the present invention, the inclusion partner nucleic acidsequence may be inserted into the chromosome of the host cell, or in avector which is preferably an expression vector. Particularly preferredas expression vectors in the present invention are well-known expressionvectors such as pAR (for lacZ), pATH (for trpE), pMAL (for malE), pGEX(for GST), or pTrxfus (for trxA). These vectors and others that may alsobe suitable are available commercially, for example from Pharmacia(Piscataway, N.J.). Alternatively, other well-characterized vectorsknown in the art may be used to carry out the methods of the presentinvention.

As described above, the methods of the present invention are suitablefor production of any polypeptide of any length, and are particularlysuitable for producing short polypeptides, or those that are toxic tothe host cells, which otherwise would not be expressed by the host cellsin significant quantities. Methods for isolation of nucleic acidsequences encoding a polypeptide of interest from a variety of sourcesare well-known in the art (see, e.g., Sambrook, J., et al., MolecularCloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, (1989)). Once the nucleic acid sequenceencoding the polypeptide of interest has been isolated, it isoperatively linked or fused to the modified inclusion partner nucleicacid from above, forming a vector, preferably an expression vector,comprising the gene fusion construct to be used in transforming the hostcells. Particularly preferred as a gene fusion construct is plasmidptcprl-monomer. Methods for fusion of the nucleic acid sequence encodinga polypeptide of interest to a truncated inclusion partner nucleic acidsequence, and insertion into an expression vector are routine to one ofordinary skill in the art (see, e.g., Ausubel, F. M., et al., eds.,Current Protocols in Molecular Biology, New York: John Wiley & Sons,Inc., pp. 16.4.1-16.8.14 (1994)).

Expression of Recombinant Protein as Inclusion Bodies

For use in the present invention, the gene fusion construct may beinserted into the chromosome of the host cell, or in a vector which ispreferably an expression vector. Introduction of the gene fusionconstruct into the host cell to produce a transformed host cell can beeffected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other methods. Such methods are described inmany standard laboratory manuals, such as Davis et al., Basic Methods InMolecular Biology (1986). Once transformed host cells have beenobtained, the cells may be cultivated under any physiologicallycompatible conditions of pH and temperature, in any suitable nutrientmedium containing assimilable sources of carbon, nitrogen and essentialminerals that support host cell growth. Recombinant protein-producingcultivation conditions will vary according to the type of vector used totransform the host cells. For example, certain expression vectorscomprise regulatory regions which require cell growth at certaintemperatures, or addition of certain chemicals or inducing agents to thecell growth medium, to initiate the gene expression resulting in theproduction of the recombinant polypeptide. Thus, the term “recombinantpolypeptide-producing conditions,” as used herein, is not meant to belimited to any one set of cultivation conditions. Appropriate culturemedia and conditions for the above-described host cells and vectors arewell-known in the art.

It has been unexpectedly found in the present invention that cultivatingthe host cells transformed with the gene fusion constructs providedherein will result in the production of the recombinant polypeptide ofinterest as inclusion bodies. Thus, routine recombinantpolypeptide-producing conditions may therefore be considered to favorproduction of the recombinant polypeptide as inclusion bodies in thehost cell, and may be used to produce recombinant polypeptides asinclusion bodies according to the present invention; the use of unusualculture conditions or undue experimentation are not required.

Isolation and Purification of Recombinant Polypeptide

As is well-known to one of ordinary skill in the art, methods for theproduction of polypeptides by recombinant DNA techniques typically aredesigned to minimize the production of the polypeptides in inclusionbodies in the host cells, due to perceived and real difficulties inisolating the polypeptides from the inclusion bodies. According to thepresent invention, however, the production of a recombinant polypeptidein inclusion bodies may be used advantageously to provide for the rapidisolation and purification of the polypeptide.

Following its production as inclusion bodies in the host cells, the genefusion product comprising the polypeptide of interest may be isolated byseveral techniques. To liberate the inclusion bodies from the hostcells, the cells must be lysed or ruptured. This lysis may beaccomplished by contacting the cells with a hypotonic solution, bytreatment with a cell wall-disrupting enzyme such as lysozyme, bysonication, by treatment with high pressure, or by a combination of theabove methods. Other methods of bacterial cell disruption and lysis thatare known to one of ordinary skill may also be used. Preferably,bacterial cells are ruptured by treatment with lysozyme followed bysonication; such treatment will yield a mixture of cellular debriscomprising the inclusion bodies, which are not disrupted by thistreatment.

Following disruption, the inclusion bodies are separated from thecellular debris by any technique suitable for separation of particles incomplex mixtures. Preferred such techniques include centrifugation, orthe use of an automated particle separator such as those commerciallyavailable from, for example, Coulter Electronics (Hialeah, Fla.). Mostpreferred for isolating inclusion bodies is centrifugation. By thepresent invention, inclusion bodies may be isolated by centrifuging thecellular debris mixture from above at about 1-25,000×g, preferably atabout 100-20,000×g, more preferably at about 5,000-15,000×g, and mostpreferably at about 10,000×g. Preferably, centrifugation is conducted atabout 4°-10° C. for about 15-60 minutes, most preferably at about 4° C.for about 30 minutes. Following centrifugation, the cellular debriscontained in the supernatant is removed from the pelleted inclusionbodies and the pellet used for purification of the recombinantpolypeptide of interest.

In preparation for purification, the gene fusion product contained inthe inclusion bodies, comprising the recombinant polypeptide, issolubilized. Solubilization is preferably accomplished by treatment witha denaturing agent, preferably guanidinium hydrochloride or urea, andmost preferably about 8M urea.

Prior to, during or following solubilization of the inclusion bodies,the recombinant polypeptide of interest may optionally be cleaved fromthe inclusion partner protein by techniques that are well-described inthe art (see, e.g., Ausubel, F. M., et al., eds., Current Protocols inMolecular Biology, New York: John Wiley & Sons, Inc., pp. 16.4.5-16.4.17(1994)). It will be understood by one of ordinary skill, however, thatproduction of recombinant polypeptides by the present invention does notnecessarily require such cleavage. This cleavage may be accomplished bya chemical cleavage method, for example by contacting the inclusionbodies with a polypeptide-releasing amount of a chemical cleavage agentunder conditions favoring the release of the polypeptide from theinclusion bodies. Preferred chemical cleavage agents include cyanogenbromide, hydroxylamine, or low pH solutions (acid hydrolysis).Alternatively, and more preferably, cleavage of the inclusion partnerprotein is accomplished by an enzymatic cleavage method, preferably bycontacting the inclusion bodies with a polypeptide-releasing amount ofan enzymatic cleavage agent under conditions favoring the release of thepolypeptide from the inclusion bodies. Preferred enzymatic cleavageagents include factor Xa (for gene fusion products comprising a malE orGST inclusion partner) or thrombin (for gene fusion products comprisinga GST inclusion partner), and particularly preferred is enterokinase(for gene fusion products comprising a trxA inclusion partner). Thesechemical and enzymatic cleavage methods are preferably carried out underconditions favoring the release of the polypeptide from the inclusionbodies, which are well-known to one of ordinary skill in the art (see,e.g., Ausubel, F. M., et al., eds., Current Protocols in MolecularBiology, New York: John Wiley & Sons, Inc., pp. 16.4.5-16.4.17 (1994)).Such release may result in solubilization of the peptide of interest andthus further solubilization may be unnecessary. Alternatively, cleavageof the inclusion partner protein from the polypeptide may be facilitatedduring the formation of the gene fusion construct. In such a scheme, anucleic acid sequence encoding a protein-specific cleavage site may beplaced between the nucleic acid sequence encoding the inclusion partnerprotein (such as the modified thioredoxin) and the recombinantpolypeptide in the gene fusion construct; upon expression of the fusionprotein as inclusion bodies in the host cells, the recombinantpolypeptide may then be isolated by treating the inclusion bodies withan enzyme (such as thrombin or enterokinase) or a chemical (such ascyanogen bromide) that specifically recognizes and cleaves at theprotein-specific cleavage site. Cleavage of the inclusion partnerprotein may alternatively be performed during or following any of thesubsequent steps described below.

After solubilization, the gene fusion product or the cleaved recombinantpolypeptide may be refolded by dialysis to remove the denaturing agent.Dialysis is preferably performed for about 18-48 hours at about 4° C.against an isotonic buffered salt solution.

Following solubilization and optional refolding, the gene fusion productor cleaved recombinant polypeptide may be purified by any of a varietyof protein purification techniques that are well-known to one ofordinary skill in the art. Suitable techniques for purification include,but are not limited to, ammonium sulfate or ethanol precipitation, acidextraction, electrophoresis, immunoadsorption, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, immunoaffinity chromatography,size exclusion chromatography, liquid chromatography (LC), highperformance LC (HPLC), fast performance LC (FPLC), hydroxylapatitechromatography and lectin chromatography. Most preferably LC, HPLC orFPLC is employed for purification.

As described above, any recombinant polypeptide may be produced andisolated from host cells by the methods of the present invention. Inparticular, it is possible to produce recombinant thioredoxin by thesemethods. In such a scheme, thioredoxin, or a fragment thereof, may beproduced by a series of steps comprising (a) modifying the thioredoxingene, (b) transferring the modified thioredoxin gene to a host cell, and(c) culturing the host cell under conditions favoring production ofthioredoxin as inclusion bodies in the host cell. These modification,transfer and culture steps may be carried out for thioredoxin asdescribed above for production of any polypeptide, and as described inmore detail in the Examples below.

Production of Protein Molecular Weight Ladders

In another aspect, the methods of the present invention may be used toprepare a protein molecular weight ladder to be used as a molecularweight or molecular sizing standard in protein analysis techniques suchas electrophoresis. In this embodiment, described in greater detailbelow in Examples 3-9, a series of fusion proteins may be made, whereinthe inclusion partner protein is linked to one or more recombinantpolypeptides or fragments thereof. For example, a nucleic acid moleculeencoding a modified thioredoxin inclusion partner protein may beinserted into a vector, preferably an expression vector, to form afusion vector such as plasmid pTrxA-concat (FIG. 4; SEQ ID NO:8). Thisvector may then be linked to single or multiple fragments of arecombinant polypeptide such as thioredoxin (FIG. 5; SEQ ID NO:11), E.coli Dead-Box protein (FIG. 6; SEQ ID NO:14), KpnI methylase (FIG. 7;SEQ ID NO:17) or 264-bp modified T4 gene 32 protein (FIG. 1; SEQ IDNO:1), each of a chosen size (e.g., 5 kD or 10 kD). After insertion ofthe nucleic acid molecule or vector into the host cell (i.e.,transformation of the host cell), the polypeptides may then be producedby expression of the nucleic acid molecules in the host cells. It willbe obvious to one of ordinary skill in the art that several expressionscenarios are possible. For example, the methods of the presentinvention may be used to produce a nucleic acid molecule encoding aplurality of the polypeptides forming the molecular weight ladder, or toproduce multiple nucleic acid molecules each of which encodes adifferent molecular weight polypeptide of the ladder. Host cells maythen be transformed with a nucleic acid encoding a plurality of suchpolypeptides, or with multiple nucleic acid molecules each encoding adifferent molecular weight polypeptide. Alternatively, multiple hostcells may be transformed, each with a single nucleic acid moleculeencoding a different polypeptide of the molecular weight ladder; in thisscenario, polypeptides produced by the host cells will be admixed toform the molecular weight ladder. In each of these scenarios, expressionof these constructs will preferably produce inclusion bodies in the hostcells comprising polypeptides from as small as 5-10 kD to as large as250-330 kD. Furthermore, the molecular weight increments of the ladderproduced by the present methods may be defined by simply altering thelength or number of copies of the recombinant polypeptide gene linked tothe inclusion partner protein gene fusion construct. Thus, it ispossible according to the present invention to produce a protein laddercomprising a collection of proteins ranging, for example, from about 510to about 300 kD, preferably from about 5 kD to about 250 kD, and morepreferably from about 10 kD to about 220 kD, in increments of, forexample, 5 kD, 1010, 2010, 25 kD, 50 kD, 100 kD or larger. Of course, itwill be understood by one of ordinary skill that other molecular weightor sizing increments may be more suitable for certain applications, andmay be prepared by only minor modifications of the present methods (suchas by increasing or decreasing the length of the gene encoding the fusedrecombinant polypeptide as described above); such methods andcompositions may thus be provided without departing from the scope ofthe present invention or any embodiment thereof.

In a preferred embodiment, the protein molecular weight ladders preparedas described above may be unstained, or may be prestained with one ormore protein-binding dyes to facilitate the use of the ladders intechniques requiring prestained protein ladders such as Westernblotting. According to the invention, any of a number of protein-bindingdyes may be used to stain proteins or the molecular weight ladders ofthe invention, to produce the prestained ladders of the invention. Anydye that binds covalently to one or more of the ladder proteins may beused, including visible dyes (chromophores), fluorescent dyes(fluorophores), phosphorescent dyes (phosphors) and the like. Preferreddyes in this regard include, but are not limited to, remazol brilliantblue R (RBBR), eosin isothiocyanate, malachite green isothiocyanate,reactive orange (also known as procion yellow), procion red, fluoresceinisothiocyanate, rhodamine isothiocyanate, eosin iodoacetamide, reactiveblack 5, Remasol brilliant violet 5R, reactive orange 14, and the like.Particularly preferred for use in the present methods are RBBR, eosinisothiocyanate and malachite green isothiocyanate. These and other dyesthat may be used in the present methods are available commercially, forexample from Sigma/Aldrich (St. Louis, Mo.) and Molecular Probes(Eugene, Oreg.).

According to the invention, prestained molecular weight ladders may beproduced by incubating one or more of the ladder proteins, which may benaturally occurring or produced recombinantly as directed above, withone or more of the above-noted dyes in a buffered aqueous solution underconditions of controlled temperature, time, and solution pH. Preferably,the ladder proteins are suspended in a buffered aqueous solution (suchas a Tris-, phosphate-, carbonate-, or HEPES-buffered saline solutioncomprising NaCl at about 10-300 mM, preferably at about 50-200 mM) at aconcentration of about 0.1 to about 25, about 0.1 to 10, or about 0.5 to10 A₂₈₀ units/ml, more preferably at a concentration of about 1-10,about 1-5, or about 1-4 A₂₈₀ units/ml. To the solution of protein(s),one or more of the above dyes may be added at concentration ranges thatare specific for each dye, typically in the range of about 0.2 mg/ml toabout 1000 mg/ml, about 0.2 mg/ml to about 500 mg/ml, about 0.2 mg/ml toabout 100 mg/ml, about 5 mg/ml to about 200 mg/ml, about 10 mg/ml toabout 200 mg/ml, or about 10 mg/ml to about 100 mg/ml. For example, RBBRmay be added to the protein solution at a final concentration of about0.3-50 mg/ml, preferably at about 5-40 mg/ml, and more preferably about10-30 mg/ml. Eosin isothiocyanate may be added to the protein solutionat a final concentration of about 1-30 mg/ml, and more preferably about7-10 mg/ml. Malachite green isothiocyanate may be added to the proteinsolution at a final concentration of about 5-30 mg/ml, more preferablyabout 20-30 mg/ml. Optimal concentrations for other dyes that may beused to stain the proteins and molecular weight ladders according to thepresent methods may be determined by one of ordinary skill without undueexperimentation, using the above-noted concentration ranges asguidelines.

The staining of the protein ladders should also be conducted underconditions of controlled solution pH and/or temperature. Preferably, thesolution is buffered to a pH (measured at about room temperature (i.e.,about 20° C.-25° C.) prior to addition of the proteins, ladders, anddye(s)) of about 7-12, about 7-11, about 8-11, or about 8.5-10.5,preferably about 7.2-9.7, more preferably about 8.2-9.7, and mostpreferably about 9.2-9.7. During the staining reaction, the solutionsshould be incubated at a temperature of about 4° C. to about 90° C.,about 4° C. to about 80° C., preferably at about room temperature (i.e.,about 20° C.-25° C.) to about 80° C., about 35° C. to about 80° C.,about 40° C. to about 75° C., about 45° C. to about 70° C., about 45° C.to about 70° C., about 50° C. to about 70° C., about 45° C. to about 65°C., more preferably at a temperature of about 50° C. to about 60° C.,and most preferably at a temperature of about 50° C. For production ofstained proteins or ladders, the protein-dye solutions should beincubated under the above-noted conditions for about 4-48 hours,preferably about 6-48 hours, more preferably about 4-24 hours, about6-24 hours, about 8-24 hours, about 10-24 hours, about 12-24 hours, andmost preferably about 12-18 hours (i.e., “overnight,” as that term willbe understood by the skilled artisan). Following incubation, the stainedprotein(s) or molecular weight ladders may be isolated from unconjugateddye and any other impurities by a variety of art-known methods (e.g.,dialysis, chromatography, gel diffusion, etc.), and may be stored insolution at −70° C. to 4° C., or they may be lyophilized and stored at−70° C. to room temperature (i.e., about 20° C.-25° C.) until use.

In a particularly preferred such embodiment, the prestained proteinladder may comprise a collection of protein molecular weight markersthat are evenly spaced on SDS-PAGE and that further comprise a referenceprotein band stained with a different color from the other bands, toallow easy orientation of all of the bands on the gel. For example, sucha prestained ladder preparation may comprise a collection of bandswherein one band (a reference band) is stained with eosin isothiocyanateand the remaining bands are stained with RBBR. Such an approach isdescribed in full detail in Example 4 below.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Materials and Methods

The following materials and methods were generally used in the examples,unless otherwise specified in a particular example.

Materials

All enzymes including restriction enzymes, T4 DNA ligase, Tag DNApolymerase and thermosensitive alkaline phosphatase were obtained fromLife Technologies, Inc. (LTI; Rockville, Md.) unless otherwise stated.The E. coli expression vector pTrc99A was obtained from Pharmacia(Piscataway, N.J.); pTrxfus was from Genetics Institute (Cambridge,Mass.) or Invitrogen (San Diego, Calif.), pRE1 was obtained from Dr.McKenney (Reddy et al., Nucl. Acids Res. 17:10473-10488 (1989)).Toyo-Pearl AF chelate-650M resin was purchased from TosoHaas(Montgomeryville, Pa.). The E. coli expression host DH10B with orwithout pRK248cl (tetracycline^(r)) and Stb12 were from LifeTechnologies, Inc. (Rockville, Md.).

Remasol brilliant blue R (RBBR) and malachite green were obtained fromAldrich and eosin isothiocyanate was obtained from Molecular Probes.Bovine aprotinin was obtained from Bayer. The cloned proteins werepurified, as fully described below, from E. coli strains which containedplasmids coding for eight proteins ranging in size from 10 kDa to 160kDa. The buffer components were all reagent grade or higher. Thediafiltration unit was a Filtron Mini Ultrasette equipped with an Omega3000 molecular weight cutoff membrane. G-25 medium resin was obtainedfrom Pharmacia. Other reagents and components were obtained fromcommercial laboratory supply sources that will be familiar to theskilled artisan.

Recombinant DNA Construction

A 264 by (SEQ ID NO:1) AvaI fragment was derived from pPrL2107 (U.S.Pat. No. 5,449,758). This fragment is a part of T4 gene 32 protein whichhas been highly modified. This AvaI fragment was used to make multimers(see U.S. Pat. No. 5,449,758) in pPrL2001. A clone with 12 inserts wasselected and was designated as pPrl 2738. The cloned fragment wasrecloned in pTrc99A as follows: the recombinant plasmid was digestedwith EcoRI and blunt ended with Klenow fragment in the presence ofdNTPs; digested with NcoI and the fragment was purified by GENE CLEAN™(Bio101). The vector, pTrc99A, was digested with SalI, blunt ended withKlenow fragment, and digested with NcoI. The gel purified vector wasligated with the gel purified fragment and transformed into DH10B. Theclone was designated as pTrcprl.

To make a monomer, pTrcprl was completely digested with AvaI to removemultimers and the vector was self-ligated. This construct contains a 261by (SEQ ID NO:2) fragment with a single AvaI site and was expected toproduce a 10 kD protein. The plasmid was designated as pTrcprl-monomer.

To generate clones to produce individual proteins from 20 kD to 220 kD,the 264 by AvaI fragment from pPrL2107 was used to make multimers inpTrcprl-monomer at the AvaI site. Since the 264 by fragment (SEQ IDNO:1) contains an internal methionine residue, it was altered to alysine residue by site-directed mutagenesis in order to stop anyinternal initiation. The ligation was carried out at room temperature(22° C.) by using 100:1 pmol ratio of insert and vector. Individualclones with correct inserts (two, three, four, etc.) were selected bythe size of the insert after digestion with NcoI and HindIII. Theplasmids were designated as pTrcprl20, pTrcprl30, pTrcprl40, and so on,to produce 20 kD, 30 kD, 40 kD, etc. proteins respectively.

A clone was also generated which contained a part of E. coli thioredoxin(truncated) under control of the lambda pL promoter to produce a 10 kDprotein. This construct contained 86 out of 108 amino acids ofthioredoxin plus six histidine residues at the carboxy end, whichprovided a His tag to facilitate purification of the expressed protein.In alternative approaches, a maximum of 33 amino acids were deleted fromthe thioredoxin protein, producing a construct comprising 75 out of 108amino acids of thioredoxin plus the His tag. The oligonucleotides usedto clone the fragment were T CTA AGG AAA TAC TTA CAT ATG AGC G (SEQ IDNO:3) and TA TTA CTG CAG TTA GTG GTG GTG GTG GTG GTG TTC ACC GTT TTT GAACAG CAG CAG (SEQ ID NO:4). The oligonucleotides were used for PCR usingpTrxfus as a template. The PCR product was digested with NdeI and PstI(incorporated in the oligonucleotides underlined) and cloned intopTrxfus digested with NdeI and PstI. The plasmid was designated aspTrxfusprl10A (SEQ ID NO:5).

The oligonucleotides TAA TAA CCA TGG CAT ATG AGC GAT AAA ATT ATT CAC(SEQ ID NO:6) and ATT ATA CCC GAG TCC ACC ACG GAT GCC ATA TTT CGG (SEQID NO:7) with NcoI (bold, underlined and italics), NdeI (bold italics)and AvaI (bold underlined) were used to generate an alternative vectorfor making multimers as follows: The oligonucleotides were used togenerate a PCR product using pTrxfus as a template. The PCR product wasdigested with NcoI and AvaI and cloned into pTrcprl-monomer to replacethe NcoI AvaI fragment of modified T4 gene 32 gene. This plasmid wasdesignated as pTrxA-concat (SEQ ID NO:8). This construct contained aminoacids 1-75 from thioredoxin, glycine as the 76th amino acid, and aminoacids 77-90 from pTrcprl-monomer including the 6 carboxy-terminalhistidine residues. This construct was made to generate fusion proteinsby in-frame ligation of an AvaI fragment or multimers of AvaI fragmentas discussed above. To make multimers, we have used AvaI fragmentsgenerated by PCR from several test genes such as thioredoxin, DEAD-boxor KpnI methylase. The oligonucleotides used to generate an AvaIfragment from the thioredoxin gene (Hoog et al., Bioscience Reports4:917-923 (1984)) were: TAA TAA CTC GGG AGC GAT AAA ATT ATT CAC CTG (SEQID NO:9) and ATA ATA CCC GAG TTT GGT TGC CGC CAC TTC ACC (SEQ ID NO:10).The PCR product (SEQ ID NO:11) was digested with AvaI and ligated toAvaI digested and dephosphorylated pTrxA-concat using 100 pmol of insertand 1 pmol of vector. Clones with single or multiple inserts wereselected by estimating the size of the insert after digestion with NcoIand PstI. Finally, the desired inserts were cloned as NdeI-PstI fragmentinto pTrxfus. Thus, these clones will producethioredoxin-(thioredoxin)_(n) fusion proteins as 20 kD, 30 kD, 40 kD,etc. The oligonucleotides used to generate an AvaI fragment fromDEAD-box gene (Toone et al., J. Bacteriol. 173(11):3291-3302 (1991))were TAA TAA CTC GGG AAG CTG ACT AAT CCG GAA GTA G (SEQ ID NO:12) andATT ATT CCC GAG CAG TGC GCG GTA TTG ATC CAG (SEQ ID NO:13). The PCRproduct (SEQ ID NO:14) was generated using E. coli chromosome astemplate. The PCR product digested with AvaI and ligated withpTrxA-concat as above. Clones with various inserts were selected andrecloned in pTrxfus as above. These clones were designed to producethioredoxin-(DEAD-box)_(n) fusion proteins as 15 kD, 20 kD, 25 kD, etc.To make thioredoxin-KpnI methylase fusions proteins, theoligonucleotides used were GA TTA CTC GGG GCA CAT AAA ATG CTA AAA GATACA (SEQ ID NO:15) and TC TAA CCC GAG TAA GAT CGT TTT TCT TGA ACC ACC(SEQ ID NO:16). The template used for PCR was a KpnI methylase clone(Chatterjee et al., Nucleic Acids Res. 19:6505-6509 (1991)). The PCRproduct (SEQ ID NO:17) was digested with AvaI and ligated topTrxA-concat and finally, an NdeI-PstI fragment was subcloned intopTrxfus plasmid. This plasmid was generated to produce a 40 kDthioredoxin-KpnI methylase fusion protein.

Expression of Proteins

E. coli DH10B or Stb12 (LTI; Rockville, Md.) containing pTrcprlconstructs were grown at 37° C. in buffer-rich media containingampicillin at 100 μg/ml. Clones were grown to an OD₅₉₀=1.0 and inducedfor 3 hours with IPTG to a final concentration of 1 nM. Clones wereincubated at 37° C. for 3 hours. pTrxfus constructs were grown at 30° C.in buffer-rich media containing ampicillin at 100 μg/ml and tetracyclineat 15 μg/ml. Clones were grown to an OD₅₉₀=1.0 and induced by heating to42° C. for 30 minutes followed a 3 hour outgrowth at 37° C. Cells wereresuspended in 1 ml of sonication buffer (10 mM Tris-HCl 7.5, 1 mM EDTA,and 10 mM β-mercaptoethanol) followed by sonicating for 3×10 seconds.Removed 100 μl for whole cell extract. An aliquot was removed to analyzeproteins as total protein. The sonicated sample was spun for 10 minutesat 4° C. and transferred supernatant to an eppendorf tube (soluble).

Samples (total and soluble fractions) were applied to a 4-20%tris-glycine gel to examine protein.

Purification of Proteins

The cells were slurried in a 1 g:2 mL ratio of 20 mM Tris-HCl, 2 mMMgCl₂, pH 8.0 buffer. Benzonase, a recombinant endonuclease fromAmerican International Chemical, was added at a ratio of 25 units per mLof slurry. The cells were cracked with two passes through a highpressure laboratory homogenizer, Model MINI-LAB, type 7.30 VH mm fromAPV Rannie, at 12,000 psi. The sample was then spun at 10,000×g for 30minutes in an RC-5 centrifuge to pellet the inclusion bodies whileleaving most of the cell debris in the supernatant liquid. The inclusionbody pellet was then washed twice at about 20-25° C. with sterile waterusing an Ultra Torrax Tissuemizer from Tekmar to fully suspend thepellet. The pellet was then made soluble in 8M Urea, 100 mM H₂NaPO₄, 4mM imidazole, pH 8.4 buffer and loaded on to a Toyopearl AF Chelate−650M resin from TosoHaas. Before loading the sample, the resin wascharged with 3 column volumes of a 1M nickel sulfate solution, washedwith three column volumes of water and then equilibrated with threecolumn volumes of 8M urea, 100 mM H₂NaPO₄, 4 mM imidazole, pH 8.4(buffer). After loading the sample, the column was washed with 10 columnvolumes of buffer, and the protein was then eluted in a single fractionwith 8M urea, 100 mM H₂NaPO₄, pH 3.5, and precipitated via dialysisagainst water containing 5 mM EDTA; EDTA was included to chelate freenickel ions which degraded the proteins upon long-term storage. Theprecipitate was spun down at 10,000×g in an RC-5 centrifuge and thensolubilized in 1% SD S/water.

Remasol Brilliant Blue R Labeling

The 10, 15, 20, 25, 30, 40, 70, 100, and 160 kDa proteins were dilutedin 1% SDS H₂O to 8 A₂₈₀ units/ml. Each protein solution was combinedwith an equal volume of the buffer 280 mM sodium phosphate, 160 mMsodium chloride, 1%(w/v) SDS, pH 9.2, and warmed to 50° C. for 15minutes. Stock solutions of RBBR (20 mg/ml and 60 mg/ml) were preparedin 140 mM sodium phosphate, 80 mM sodium chloride, 1% (w/v) SDS, pH 9.2,vortexed, and incubated for 15 minutes at 50° C. The 10, 15, 20, 30, 70,and 160 kDa protein solutions were then individually mixed with an equalvolume of the warmed 60 mg/ml RBBR stock, vortexed, and incubated for14-18 hours at 50° C. The 40 and 100kDa protein solutions were mixedindividually with an equal volume of the warmed 20 mg/ml RBBR stock,vortexed, and incubated for 14-18 hours at 50° C. Following incubation,solutions were removed from the waterbath, allowed to equilibrate toroom temperature, filtered through a 0.8 μm cameo filter to remove anyresidual particulate, and stored at 4° C. until use.

To provide a low molecular weight standard, aprotinin was prepared at aconcentration of 11 mg/ml in 140 mM sodium phosphate, 80 mM sodiumchloride, 3%(w/v) SDS. The solution was gently vortexed and warmed to50° C. for 15 minutes. After warming, 5.7 mg of RBBR/ml was added to thesolution, and the solution was then mixed and incubated for 40 minutesat 70° C. β-mercaptoethanol was then added (6.9 μl/ml), and the solutionwas again mixed and incubated for an additional 10 minutes at 70° C.,after which 9.1 μl/ml of acrylonitrile was added. The solution wasmixed, incubated for 30 minutes at room temperature, and then filteredthrough a 0.8 μm cameo filter cartridge and stored at 4° C.

Eosin Isothiocyanate Labeling of the 50 kDa Protein

The 8 A₂₈₀/ml 50 kDa stock in 1% SDS/H₂O was combined with an equalvolume of 280 mM sodium phosphate, 160 mM sodium chloride, 1% (w/v) SDS,pH 9.7 buffer, mixed, and warmed for 15 minutes at 50° C. A 70 mg/mlstock of eosin isothiocyanate (EITC) was prepared in dimethylformamideimmediately before use, and a sufficient volume of this solution wasadded to the 50 kDa protein solution to achieve a final concentration of7 mg/ml EITC. The solution was then mixed, incubated at 50° C. for 14-18hours in the dark, equilibrated to room temperature, filtered through a0.8 nm cameo filter cartridge, and stored in the dark at 4° C.

Preparation of Final Mixture

The quality of the labeling was assessed by running each protein stockon SDS-PAGE (Laemmli, E. K., Nature 227:680-685 (1970)), then preparinga mix of all of the proteins so that the amounts of the proteins producebands which appear visually to have equivalent intensities. The mix wasthen diafiltered using a 3,000 molecular weight cutoff miniultrasetteunit with 10% glycerol, 50 mM TRIS, 5 mM EDTA (to enhance stability ofthe preparation upon storage), pH 6.8 buffer. A Sephadex G-25 column wasused to remove a small amount of unreacted dye which remained afterdiafiltration. The Sephadex column that was equilibrated with 50 mMTris, 1% (w/v) SDS, 5 mM EDTA, pH 6.8, and the column size was 15 timesthe volume of the sample. SDS-PAGE was carried out on the final mixtureto assess the quality of the mix.

EXAMPLE 1 Cloning and Expression of a 10 kD Protein

The initial objective was to make a protein molecular weight standardranging from 10 kD-250 kD with 10 kD increments. Therefore, a modifiedportion of T4 gene 32 protein containing 87 amino acids (261 bp) thatincludes 6 histidine residues at the carboxy end (FIG. 1; SEQ ID NO:1)(see U.S. Pat. No. 5,449,758) was used. The fragment was cloned andexpressed to produce a 10 kD protein. The clone was designated asplasmid pTrcprl-monomer. This construct contains an unique AvaI site(CTCGGG) to generate multimers of any given AvaI fragment with CTCGGGsequence. When digested with AvaI, the clone generates a TCGG overhang.Thus, when a DNA fragment having a TCGG overhang at both ends is ligatedwith AvaI-digested pTrcprl-monomer, the fragment will be ligated only inone direction (head-to-tail). Using proper ligation condition, it waspossible to generate multimers of a desired fragment. For example, if asingle AvaI fragment (264 bp) is ligated to pTrcprl-monomer it willproduce a 20 kD protein; if two fragments are ligated, it will produce a30 kD protein; and so on.

As noted above, the pTrcprl-monomer was designed to produce a 10 kDprotein. However, the induced or uninduced cultures did not produce any10 kD protein. Interestingly, the larger clones with multiple insertsproduced proteins of expected sizes. The level of expression, however,varied among the clones depending on the number of inserts. The level ofexpression of various proteins (in kD) was as follows:20<30<40<50<60<70≅80≅90≅100≅120>160>180>200>220>250.

To obtain a protein of low molecular weight for use in the production ofmolecular weight standards, natural proteins close to 10 kD in size thatare well-expressed in E.coli were sought. One such protein isthioredoxin, which consists of 108 amino acids and which has a molecularmass of 12 kD (Lunn et al., J. Biol. Chem. 259:10469-10474 (1984); Hooget al., BioSci Rep. 4:917-923 (1984)). Thioredoxin is ubiquitouslydistributed, and is present in an unusually high concentration per cell(10,000-20,000 molecules/cell). In addition, it has also been shown thatwhen overexpressed, it represents almost 40% of the total solublecellular protein (Lunn et al., Id.). It thus appeared that thioredoxinmight be a suitable candidate for the production of a 10 kD protein if a2 kD portion was deleted from its carboxy terminus. One such construct(pTrxfusprl10A) which produced a 10 kD protein contained: a) amino acids1-85 from thioredoxin; b) a substitution of glutamic acid for valine atamino acid position number 86; and c) histidine residues at positions87-92. This truncated thioredoxin construct produced about 20-30% of thetotal cellular protein. However, unlike full length thioredoxin thisconstruct produced almost all of the induced protein as inclusionbodies. In other experiments, 33 of the 108 amino acids were deletedfrom the thioredoxin carboxy terminus; such constructs also producedalmost all of the induced protein as inclusion bodies upon expression inthe host cells. It may also be possible to produce polypeptides by thepresent methods using truncated thioredoxin in which 2-22 amino acidshave been deleted from the carboxy terminus, or in which 33-50 aminoacids have been deleted, as described above. Following expression of theprotein in the host cells, the inclusion bodies were easily isolatedfrom the host cells by centrifugation.

EXAMPLE 2 Cloning of Fusion Proteins Larger than 10 kD

In order to make fusion protein with truncated thioredoxin, a vectorcontaining a unique AvaI site, used to make concatamers, was developed;this vector was designated pTrxA-concat (FIG. 4; SEQ ID NO:8). Usingthis vector, a series of fusion proteins has been made linking thevector to single or multiple fragments of thioredoxin (FIG. 5; SEQ IDNO:11) (thioredoxin-thioredoxin fusion), E. coli DEAD-box protein (FIG.6; SEQ ID NO:14), KpnI methylase (FIG. 7; SEQ ID NO:17), or 264-bpmodified T4 gene 32 protein (FIG. 1; SEQ ID NO:1) (See U.S. Pat. No.5,449,758).

Specifically, fusion proteins of the indicated molecular weights weremade by ligating pTrxA-concat to one or more copies of the nucleic acidmolecules encoding 10 kD fragments of truncated E. coli thioredoxin(ΔtrxA) or T4 gene 32 protein, or 5 kD fragments of E. coli Dead-Boxprotein or KpnI methylase, as shown in Table 1. In all cases, the fusionprotein was expressed as inclusion bodies. Thus, using the methods ofthe present invention, polypeptides ranging in size from 10 kD to 220kD, in 5-10 kD increments, have been efficiently produced as inclusionbodies.

TABLE 1 Scheme for Production of Fusion Proteins over a Range ofMolecular Weights. Number of Copies of Gene Ligated to Vector 10 kD T4Molecular 5 kD Dead- Gene 32 5 kD KpnI Weight (kD) 10 kD ΔtrxA BoxProtein Protein Methylase 10 1 0 0 0 15 1 1 0 0 20 1 2 0 0 25 1 0 0 3 303 0 0 0 40 1 0 3 0 50 1 0 4 0 50 5 0 0 0 60 6 0 0 0 70 1 0 6 0 80 8 0 00 90 9 0 0 0 100 1 0 9 0 120 1 0 11 0 160 1 0 15 0 220 1 0 21 0

EXAMPLE 3 Production of Unstained Protein Molecular Weight Ladders

To demonstrate its utility, the present system was used to makeunstained protein molecular weight ladders. Purified proteins ofdiffering sizes, made as described in Example 2, were mixed andseparated by SDS-PAGE, and the banding patterns in various acrylamideconcentrations was observed.

The results of these experiments showed the combination of proteinsallowing the best resolution over a range of acrylamide concentrationsto be a mixture of 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 40 kD, 50 kD, 60kD, 70 kD, 80 kD, 90 kD, 100 kD, 120 kD, 160 kD and 220 kD proteins.These results demonstrate that the methods of the present invention areuseful in making proteins of various size ranges, and may be used toproduce compositions comprising protein molecular weight ladders.

EXAMPLE 4 Production of Prestained Protein Molecular Weight Ladders

Prestained protein molecular weight ladders have been commerciallyavailable for more than 10 years in two distinct forms. In one form, allof the proteins are stained with a single dye, and in the second formeach protein is stained with different dyes (a multicolored version).These two distinct forms have several advantages and disadvantages.Single color markers, for example, are typically prepared in a way thatresults in relatively sharp bands in SDS-PAGE compared to multicoloredmarkers, but directly relating each band to its corresponding molecularweight can be difficult since the only point of reference in singlecolored prestained markers is the highest or lowest molecular weightband. If either of these reference bands disappears during storage, oris not resolved on the gel, the absence of a point of reference makesidentification of the remaining bands difficult. Similarly,determination of the identity of the bands in multicolored markers iseasy since each band is a distinct color, but some of the bands are verybroad in SDS-PAGE and some of the colors are difficult to detectvisually.

In the present invention, a new set of prestained protein molecularweight markers has been developed. These markers are evenly spaced onSDS-PAGE and comprise a reference band of a different color from theother bands to allow easy orientation of all of the marker bands. Asingle dye was used to stain all but one protein (the referenceprotein), which was stained with a second dye. Staining protocols weredeveloped to produce prestained proteins that gave sharp bands onSDS-PAGE using both dyes, in contrast to the wide variations in bandsharpness common in multicolored markers. The protein stained with thesecond dye serves as a point of reference within the marker facilitatingthe identification of the prestained marker bands. A number of dyes weretested including Remasol brilliant blue R (RBBR), eosin isothiocyanate,procion red, reactive orange (also known as procion yellow), eosiniodoacetamide, reactive black 5, Remasol brilliant violet 5R, reactiveorange 14, rhodamine isothiocyanate, and malachite green isothiocyanate.Labeling and storage conditions of each protein component was optimizedin terms of buffer pH and ionic composition, dye concentration, proteinconcentration, temperature of labeling, and incubation time duringlabeling, as described in detail above in the Materials and Methodssection.

A number of different cloned proteins were labeled with RBBR and thenrun on SDS-PAGE to determine the effectiveness of the labeling procedureand to determine the apparent molecular weights of the labeled proteins.As shown in FIG. 8, the most intense staining and most even spacing onSDS-PAGE were achieved with the combination of 10, 15, 20, 30, 40, 50,70, 100, and 160 kDa proteins. The 50 kDa protein was labeled with eosinisothiocyanate so that a highlight or reference band could beincorporated into the marker ladder. This bright pink band waspositioned as the fourth band from the top and the sixth band from thebottom (not including the aprotinin standard which is not part of theladder composition), providing an easy point of reference to determinethe molecular weight of a particular band. These results demonstratethat the present methods may be used to produce molecular weight markersets that are easily prestained.

EXAMPLE 5 Production of Molecular Weight Markers for use in Blotting

It would be useful to have molecular weight markers that can not only beused to determine molecular weight after coomassie blue staining in agel, but that also could be used as molecular weight markers followingmembrane transfer techniques such as western blotting. The molecularweight markers produced by the present methods can be used for both ofthese applications. As described above, each of the protein componentsin the unstained molecular weight marker mixture contains six histidineresidues at the carboxy terminus. Since these six histidine residuesinteract with Ni⁺⁺ (Hochuli, E., and Piesecki, S., Methods: A Companionto Methods in Enzymology 4:68-72 (1992), a Ni-NTA-alkaline phosphataseconjugate along with nitroblue tetrazolium chloride(NBT)/5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) can beused to detect and identify the protein components in a Western Blot.

EXAMPLE 6 Production of Small or Toxic Proteins

The present system is particularly useful to express proteins that aredifficult to express in E. coli, whether this difficulty is due to (1)smaller size of the protein (in general, smaller proteins are expressedat lower levels in E. coli); (2) toxicity of the heterologous protein tothe E. coli host; (3) inefficient translation in E. coli; or (4)instability of the mRNA. As shown in the examples above, the presentmethods allow efficient production of heterologous proteins as inclusionbodies. Since inclusion bodies are an isolated form of the expressedprotein, this system may be used to produce toxic proteins or smallpeptides which otherwise might not be produced in E. coli or otherbacterial host cells. Using this system, peptides of 5 kD and smallerhave been produced. The system can also be used to produce polypeptides250 kD in size or larger.

EXAMPLE 7 Optimization of Conditions for Labeling Protein Ladders WithRemasol Brilliant Blue R (RBBR)

In general, chemical modification or conjugation of a protein (e.g.,labeling it with a dye) involves the reaction of functionally reactivegroups on the protein with corresponding functionally reactive groups onthe label. Reactive groups able to couple with amine-containingmolecules, such as proteins containing lysine residues, are by far themost common functional groups present on cross-linking reagents such asdyes. An amine-coupling process therefore can be used to conjugatenearly all proteins or peptides with other molecules.

In the present invention, a variety of dyes, including remazol brilliantblue R (RBBR) and eosin isothiocyanate, have been used as couplingreagents for labeling of molecular weight ladder proteins. These dyesprimarily react with nucleophilic amine groups on the proteins to yieldstable protein-dye complexes. The dyes can also react, to some extent,with other nucleophiles on the target proteins, such as sulfhydrylgroups and phenolate ions of tyrosine side chains.

To produce the most useful (i.e., highest color intensity and bandsharpness) and consistent (i.e., lowest variability in band mobilitybetween batches) prestained protein ladders, it is essential to optimizethe staining protocol. The band intensity will depend upon the amount ofprimary amine groups present in the protein being conjugated with thedye, while the band sharpness will depend upon how many of the availableprimary amines in the target protein have been conjugated with the dye.Each of these variables will be dependent upon the environmentalconditions to which the protein and dye are subjected duringconjugation. Thus, the extent of conjugation of the target markerproteins with dye will depend upon a number of conditions duringstaining, including concentrations of the protein and dye in solution,pH of the solution, ionic conditions, temperature, and duration ofincubation of protein with the dye.

Thus, one object of the present invention is to develop conditions tostain protein molecular weight markers that will provide molecularweight ladders with bands that resolve as intensely, as homogeneously,and as consistently as possible. To meet this objective, the labelingconditions in terms of concentrations of the protein and dye insolution, pH of the solution (adjusted prior to addition of protein(s)and dye(s)), and temperature and time of incubation, were optimized.

To minimize volumes in which the reactions were carried out, variedconcentrations of proteins were examined. Proteins produced as describedabove were prepared in solutions at 1, 2, and 4 A₂₈₀ units/ml, andlabeled with RBBR as generally described above in the Materials andMethods section, and then separated on 4-20% SDS-PAGE gradient gels asabove in Example 4. Intensity of staining was then determined bydensitometry and by eyesight. In general, protein concentrations of 1and 2 A₂₈₀ units/ml produced signals of similar intensity, whilelabeling intensity was lower at 4 A₂₈₀ units/ml.

In analogous experiments, the optimal concentration of RBBR wasdetermined by staining proteins with RBBR at a range of concentrationsfrom 0.3 to 30 mg/ml. In general, 10 mg/ml RBBR was found to provide themost intensely staining, homogeneous product when proteins of 40 kD and100 kD were labeled, while proteins of 10 kD, 15 kD, 20 kD, 30 kD, 45kD, 50 kD, 60 kD, 70 kD, and 160 kD, were optimally labeled by 30 mg/nilof RBBR. These results suggest that a range of about 5-50 mg/ml, or10-30 mg/ml, of RBBR may be optimal for labeling of protein molecularweight ladders consisting of proteins of varying molecular weights.

In preliminary experiments designed to determine optimal temperature andtime periods for labeling, incubation of the proteins in solutionwithout dye indicated that the proteins tended to degrade when incubatedat 70° C. for 0.5-1 hour, and when incubated at 60° C. for about 2-5hours. When incubated overnight (12-24 hours) at about 50° C. or lower,however, the proteins appeared to be stable (i.e., to resist degradationin solution). Upon staining the protein solutions with RBBR at theabove-noted concentration ranges, the most homogeneous and intense bands(i.e., sharpest and brightest banding patterns) were observed in proteinpreparations stained with RBBR overnight at 50° C. Shorter incubationperiods (e.g., 4 to 8 hours) produced more heterogeneous (i.e., morediffuse and less intense) banding patterns, particularly with lowermolecular weight proteins (10 kD to 60 kD). Overnight incubations atroom temperature (about 20° C. to 25° C.) and at 37° C. did not producesatisfactory results; protein ladder preparations stained with RBBRunder these conditions resulted in ladders demonstrating even morediffuse and less intense banding patterns. These results indicate thatovernight staining of proteins at 50° C. is optimal for producingprotein molecular weight ladders prestained with RBBR.

Finally, to determine pH optima for staining of the ladder preparationswith RBBR, proteins were labeled with RBBR as above in solutionsbuffered to pHs ranging from about 7.2 to about 9.7. Ideal results(i.e., most intensely staining and least diffuse banding patterns) wereobserved in solutions buffered to pH 9.2. It is to be noted, however,that the pH of the final staining mixture was about 8.3 (after additionof protein and dye), while the optimal initial pH of the buffer to whichprotein and dye were added was about 9.2.

Taken together, these results suggest that the optimal conditions forproducing protein molecular weight ladders prestained with RBBR includea protein concentration of about 1 to 2 A₂₈₀ units/ml, a dyeconcentration of about 5 to about 50 mg/ml, a pH of about 9.0 to 9.5,and incubation of the protein and dye for about 12-24 hours at atemperature of about 50° C.

EXAMPLE 8 Optimization of Conditions for Labeling Protein Ladders WithEosin Isothiocyanate

As noted in Example 4 above, eosin labeling of a single protein band ina complex protein molecular weight ladder, particularly between theRBBR-labeled 40 kD and 70 kD bands, provides a valuable reference bandthat permits rapid and accurate identification of all of the otherprotein bands in the ladder. Therefore, the optimal conditions forlabeling 30 kD, 40 kD, 50 kD and 60 kD proteins with eosinisothiocyanate, for use in the ladders of the invention, weredetermined.

Various preparations of 30 kD, 40 kD, 50 kD and 60 kD recombinantproteins, prepared as described above in Examples 1 and 2, wereincubated with eosin isothiocyanate under the conditions of proteinconcentration, dye concentration, temperature, time, and pH, used forthe studies with RBBR in Example 7. Following labeling with eosinisothiocyanate, protein preparations were run on 4-20% SDS-PAGE.

As shown in FIGS. 9-12, the pH and temperature conditions used duringeosin labeling of 30 kD, 40 kD, 50 kD and 60 kD reference proteinsdramatically affected the intensity of staining and sharpness ofresolution of the bands on SDS-PAGE. Optimal results were obtained whenabout 2 A₂₀ units of the 50 kD protein were incubated overnight (ideallyfor about 4-16 hours) at 50° C. with a final concentration of eosinisothiocyanate of about 7 mg/ml at pH 9.2 (see FIG. 9, lane 5; FIG. 10,lane 13; FIG. 11, lane 14; and FIG. 12, lane 8), and when the 60 kDprotein was labeled under the same conditions, except at pH 9.2-9.7 (seeFIG. 9, lane 13, and FIG. 12, lane 6). Under these same conditions,however, incubation of the proteins with the commonly used dye procionred produced little or no staining of the ladder bands (see FIG. 12,lanes 1-4).

EXAMPLE 9 Optimization of Conditions for Labeling Protein Ladders WithMalachite Green Isothiocyanate

To provide reference bands and proteins ladders stained with a varietyof colors, it would be advantageous to stain the protein ladders of theinvention with a dye, such as malachite green, of different color fromRBBR and eosin. Therefore, the optimal conditions for labeling 30 kD, 40kD, 50 kD and 60 kD proteins with malachite green isothiocyanate weredetermined.

Various preparations of 30 kD, 40 kD, 50 kD and 60 kD recombinantproteins, prepared as described above in Examples 1 and 2, wereincubated with malachite green isothiocyanate under the conditions ofprotein concentration, dye concentration, temperature, time, and pH,used for the studies with RBBR in Example 7 and with eosinisothiocyanate in Example 8. Following labeling with malachite greenisothiocyanate, protein preparations were run on 4-20% SDS-PAGE.

As shown in FIG. 13, the pH conditions used during malachite greenlabeling of 40 kD, 50 kD and 60 kD reference proteins dramaticallyaffected the intensity of staining and sharpness of resolution of thebands on SDS-PAGE. Optimal results were obtained when about 1-2 A₂₈₀units of the proteins were incubated overnight (ideally for about 16hours) at room temperature with a final concentration of malachite greenisothiocyanate of about 20-30 mg/ml at pH 9.2 (see FIG. 13, lanes 2, 4,6 and 8).

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1-68. (canceled)
 69. A method for preparing a pre-stained proteincomposition comprising a homogenous stained population of a protein, byincubating the protein with a dye, the molecules of the homogenousstained protein population having the same mobility during gelelectrophoresis, and the molecules of the population having the propertythat, if they were further incubated with the dye, their mobility duringgel electrophoresis would remain unchanged.
 70. A pre-stained proteincomposition, comprising a homogenous stained population of a protein,the molecules of the homogenous stained protein population having thesame mobility during gel electrophoresis, and the molecules of thepopulation having the property that, if they were further incubated withthe dye, their mobility during gel electrophoresis would remainunchanged.
 71. The composition of claim 70, when comprised in anelectrophoresis gel.
 72. The use as a protein ladder composition of aplurality of homogeneous stained protein populations, the molecules ofeach population having the same mobility during gel electrophoresis, themolecules of the population having the property that, if they werefurther incubated with the dye, their mobility during gelelectrophoresis would remain unchanged, and the protein of each of theplurality of populations having a different molecular weight from theprotein of each other population.
 73. The use of claim 72, wherein allthe populations except for a single population are stained with the samedye.
 74. A method of separating proteins by weight by gelelectrophoresis involving a molecular weight ladder compositionaccording to claim 69.